title: Kinetics of the reactions of the Escherichia coli molecular chaperone DnaK with ATP: evidence that a three-step reaction precedes ATP hydrolysis.
abstract: The mechanism of the ATPase cycle of the 70-kDa Escherichia coli molecular chaperone DnaK was investigated by following ATP-induced changes in the tryptophan fluorescence of DnaK. Three steps in the cycle were investigated. (i) Stopped-flow experiments revealed that ATP induces a biphasic reduction in the tryptophan fluorescence of DnaK. The rate of the fast fluorescence transition exhibited a hyperbolic dependence on the ATP concentration, with a maximum rate equal to 56 (+/- 10) s-1 at 35 degrees C, whereas the rate of the slow fluorescence transition was nearly independent of the ATP concentration (4.2 +/- 0.2 s-1). These results are consistent with the three-step sequential reaction E + ATP<-->E-ATP<-->E*-ATP<-->E**-ATP prior to DnaK-catalyzed ATP hydrolysis, where the formation of a collisional complex (E-ATP) causes no change in fluorescence but is followed by two first-order transitions that reduce the fluorescence. (ii) The kinetics of ADP replacement from preformed DnaK-ADP complexes by ATP followed simple exponential kinetics, kADP = 0.038 (+/- 0.002) s-1 at 35 degrees C. The ADP off rate was reduced approximately 10-fold by inorganic phosphate (20 mM). (iii) Single-turnover experiments ([DnaK] = [ATP] = 1 microM) revealed a slow, first-order increase in tryptophan fluorescence [k(obs) = 0.0015 (+/- 0.0001) s-1, 37 degrees C] that was identical to the rate of DnaK-catalyzed ATP hydrolysis [k(hy) = 0.0014 (+/- 0.0001) s-1, 37 degrees C]. This slow increase in fluorescence is consistent with a E**-->E conformational transition. A model for the ATPase cycle of DnaK is proposed in which ATP has two distinct functions: ATP binding to the ATPase domain triggers two conformational transitions in a chaperone molecule, and ATP hydrolysis--the slow step in the reaction cycle--reverses the transitions.
authors.affiliation: Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, Shreveport 71130-3932, USA.
authors.forename: S V
authors.initials: SV
authors.lastname: Slepenkov
authors.forename: S N
authors.initials: SN
authors.lastname: Witt
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008956:Models, Chemical; D018832:Molecular Chaperones / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D018832:Molecular Chaperones; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; C063842:dnaK protein, E coli